Sdf-1 delivery for treating ischemic tissue

ABSTRACT

A method of treating a cardiomyopathy in a subject includes administering directly to or expressing locally in a weakened, ischemic, and/or peri-infarct region of myocardial tissue of the subject an amount of SDF-1 effective to cause functional improvement in at least one of the following parameters: left ventricular volume, left ventricular area, left ventricular dimension, cardiac function, 6-minute walk test, or New York Heart Association (NYHA) functional classification.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/393,141, filed Jun. 7, 2012 as entry into the U.S. national stage ofInternational Application No. PCT/US2010/047175, filed Aug. 30, 2010,which, in turn, claims priority from U.S. Provisional Application Nos.61/237,775, filed Aug. 28, 2009, and 61/334,216, filed May 13, 2010. Thesubject matter of the foregoing applications is incorporated herein byreference in its entirety for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 23, 2012, isnamed JUVE0078.txt and is 5,914 bytes in size.

FIELD OF THE INVENTION

This application relates to SDF-1 delivery methods and compositions fortreating a cardiomyopathy and to the use of SDF-1 delivery methods andcompositions for treating an ischemic cardiomyopathy.

BACKGROUND OF THE INVENTION

Ischemia is a condition wherein the blood flow is completely obstructedor considerably reduced in localized parts of the body, resulting inanoxia, reduced supply of substrates and accumulation of metabolites.Although the extent of ischemia depends on the acuteness of vascularobstruction, its duration, tissue sensitivity to it, and developmentalextent of collateral vessels, dysfunction usually occurs in ischemicorgans or tissues, and prolonged ischemia results in atrophy,denaturation, apoptosis, and necrosis of affected tissues.

In ischemic cardiomyopathy, which are diseases that affect the coronaryartery and cause myocardial ischemia, the extent of ischemic myocardialcell injury proceeds from reversible cell damage to irreversible celldamage with increasing time of the coronary artery obstruction.

SUMMARY OF THE INVENTION

This application relates to a method of treating a cardiomyopathy in asubject. The cardiomyopathy can include, for example, cardiomyopathiesassociated with a pulmonary embolus, a venous thrombosis, a myocardialinfarction, a transient ischemic attack, a peripheral vascular disorder,atherosclerosis, and/or other myocardial injury or vascular disease. Themethod includes administering directly to or expressing locally in aweakened, ischemic, and/or peri-infarct region of myocardial tissue ofthe subject an amount of SDF-1 effective to cause functional improvementin at least one of the following parameters: left ventricular volume,left ventricular area, left ventricular dimension, cardiac function,6-minute walk test (6MWT), or New York Heart Association (NYHA)functional classification.

In an aspect of the application, the amount of SDF-1 administered to theweakened, ischemic, and/or peri-infarct region is effective to causefunctional improvement in at least one of left ventricular end systolicvolume, left ventricular ejection fraction, wall motion score index,left ventricular end diastolic length, left ventricular end systoliclength, left ventricular end diastolic area, left ventricular endsystolic area, left ventricular end diastolic volume, 6-minute walk test(6MWT), or New York Heart Association (NYHA) functional classification.In another aspect of the application, the amount of SDF-1 administeredto the weakened, ischemic, and/or peri-infarct region is effective toimprove left ventricular end systolic volume. In a further aspect of theapplication, the amount of SDF-1 administered to the weakened, ischemic,and/or peri-infarct region is effective to improve left ventricularejection fraction.

In some aspects of the application, the amount of SDF-1 administered tothe weakened, ischemic, and/or peri-infarct region is effective toimprove left ventricular end systolic volume by at least about 10%. Inother aspects of the application, the amount of SDF-1 administered tothe weakened, ischemic, and/or peri-infarct region is effective toimprove left ventricular end systolic volume by at least about 15%. Instill further aspects of the application, the amount of SDF-1administered to the weakened, ischemic, and/or peri-infarct region iseffective to improve left ventricular end systolic volume by at leastabout 10%, improve left ventricular ejection fraction by at least about10%, improve wall motion score index by at least about 5%, improve sixminute walk distance at least about 30 meters, and improve NYHA class byat least 1 class. In a further aspect of the application, the amount ofSDF-1 administered to the weakened, ischemic, and/or peri-infarct regionis effective to improve left ventricular ejection fraction by at leastabout 10%.

In another aspect of the application, the amount of SDF-1 administeredto the weakened, ischemic, and/or peri-infarct region is effective tosubstantially improve vasculogenesis of the weakened, ischemic, and/orperi-infarct region by at least about 20% based on vessel density ormeasured by myocardial perfusion imaging (e.g., SPECT or PET) with animprovement in summed rest score, summed stress score, and/or summeddifference score of at least about 10%. The SDF-1 can be administered byinjecting a solution comprising SDF-1 expressing plasmid in theweakened, ischemic, and/or peri-infarct region and expressing SDF-1 fromthe weakened, ischemic, and/or peri-infarct region. The SDF-1 can beexpressed from the weakened, ischemic, and/or peri-infarct region at anamount effective to improve left ventricular end systolic volume.

In an aspect of the application, the SDF-1 plasmid can be administeredto the weakened, ischemic, and/or peri-infarct region in multipleinjections of the solution with each injection comprising about 0.33mg/ml to about 5 mg/ml of SDF-1 plasmid solution. In one example, theSDF-1 plasmid can be administered to the weakened, ischemic, and/orperi-infarct region in at least about 10 injections. Each injectionadministered to the weakened, ischemic, and/or peri-infarct region canhave a volume of at least about 0.2 ml. The SDF-1 can be expressed inthe weakened, ischemic, and/or peri-infarct region for greater thanabout three days.

In an example application, each injection of solution comprising SDF-1expressing plasmid can have an injection volume of at least about 0.2 mland an SDF-1 plasmid concentration per injection of about 0.33 mg/ml toabout 5 mg/ml. In another aspect of the application, at least onefunctional parameter of the of the heart can be improved by injectingthe SDF-1 plasmid into the weakened, ischemic, and/or peri-infarctregion of the heart at an injection volume per site of at least about0.2 ml, in at least about 10 injection sites, and at an SDF-1 plasmidconcentration per injection of about 0.33 mg/ml to about 5 mg/ml.

In a further example, the amount of SDF-1 plasmid administered to theweakened, ischemic, and/or peri-infarct region that can improve at leastone functional parameter of the heart is greater than about 4 mg. Thevolume of solution of SDF-1 plasmid administered to the weakened,ischemic, and/or peri-infarct region that can improve at least onefunctional parameter of the heart is at least about 10 ml.

In another aspect of the application, the subject to which the SDF-1 isadministered can be a large mammal, such as a human or pig. The SDF-1plasmid can be administered to the subject by catheterization, such asintra-coronary catheterization or endo-ventricular catheterization. Themyocardial tissue of the subject can be imaged to define the area ofweakened, ischemic, and/or peri-infarct region prior to administrationof the SDF-1 plasmid, and the SDF-1 plasmid can be administered to theweakened, ischemic, and/or peri-infarct region defined by the imaging.The imaging can include at least one of echocardiography, magneticresonance imaging, coronary angiogram, electroanatomical mapping, orfluoroscopy.

The application also relates to a method of treating a myocardialinfarction in a large mammal by administering SDF-1 plasmid to theperi-infarct region of the myocardium of the mammal by catheterization,such as intra-coronary catheterization or endo-ventricularcatheterization. The SDF-1 administered by catheterization can beexpressed from the peri-infarct region at an amount effective to causefunctional improvement in at least one of the following parameters: leftventricular volume, left ventricular area, left ventricular dimension,cardiac function, 6-minute walk test (6MWT), or New York HeartAssociation (NYHA) functional classification.

In an aspect of the application, the amount of SDF-1 administered to theperi-infarct region is effective to cause functional improvement in atleast one of left ventricular end systolic volume, left ventricularejection fraction, wall motion score index, left ventricular enddiastolic length, left ventricular end systolic length, left ventricularend diastolic area, left ventricular end systolic area, left ventricularend diastolic volume, 6-minute walk test (6MWT), or New York HeartAssociation (NYHA) functional classification. In another aspect of theapplication, the amount of SDF-1 administered to the peri-infarct regionis effective to improve left ventricular end systolic volume. In afurther aspect of the application, the amount of SDF-1 administered tothe weakened, ischemic, and/or peri-infarct region is effective toimprove left ventricular ejection fraction.

In some aspects of the application, the amount of SDF-1 administered tothe peri-infarct region is effective to improve left ventricular endsystolic volume by at least about 10%. In other aspects of theapplication, the amount of SDF-1 administered to the peri-infarct regionis effective to improve left ventricular end systolic volume by at leastabout 15%. In still further aspects of the application, the amount ofSDF-1 administered to the peri-infarct region is effective to improveleft ventricular end systolic volume by at least about 10%, improve leftventricular ejection fraction by at least about 10%, improve wall motionscore index by about 5%, improve six minute walk distance at least about30 meters, or improve NYHA class by at least 1 class. In a furtheraspect of the application, the amount of SDF-1 administered to theweakened, ischemic, and/or peri-infarct region is effective to improveleft ventricular ejection fraction by at least about 10%.

In another aspect of the application, the amount of SDF-1 administeredto the peri-infarct region is effective to substantially improvevasculogenesis of the peri-infarct region by at least about 20% based onvessel density.

In an aspect of the application, the SDF-1 plasmid can be administeredto the weakened, ischemic, and/or peri-infarct region in multipleinjections of the solution with each injection comprising about 0.33mg/ml to about 5 mg/ml of SDF-1 plasmid/solution. In one example, theSDF-1 plasmid can be administered to the weakened, ischemic, and/orperi-infarct region in at least about 10 injections. Each injectionadministered to the weakened, ischemic, and/or peri-infarct region canhave a volume of at least about 0.2 ml. The SDF-1 can be expressed inthe weakened, ischemic, and/or peri-infarct region for greater thanabout three days.

In an example application, each injection of solution comprising SDF-1expressing plasmid can have an injection volume of at least about 0.2 mland an SDF-1 plasmid concentration per injection of about 0.33 mg/ml toabout 5 mg/ml. In another aspect of the application, at least onefunctional parameter of the of the heart can be improved by injectingthe SDF-1 plasmid into the weakened, ischemic, and/or peri-infarctregion of the heart at an injection volume per site of at least about0.2 ml, in at least about 10 injection sites, and at an SDF-1 plasmidconcentration per injection of about 0.33 mg/ml to about 5 mg/ml.

In a further example, the amount of SDF-1 plasmid administered to theweakened, ischemic, and/or peri-infarct region that can improve at leastone functional parameter of the heart is greater than about 4 mg. Thevolume of solution of SDF-1 plasmid administered to the weakened,ischemic, and/or peri-infarct region that can improve at least onefunctional parameter of the heart is at least about 10 ml.

The application further relates to a method of improving leftventricular end systolic volume in a large mammal after myocardialinfarction. The method includes administering SDF-1 plasmid to theperi-infarct region of the mammal by endo-ventricular catheterization.The SDF-1 can be expressed from the peri-infarct region at an amounteffective to cause functional improvement in left ventricular endsystolic volume.

In some aspects of the application, the amount of SDF-1 administered tothe peri-infarct region is effective to improve left ventricular endsystolic volume by at least about 10%. In other aspects of theapplication, the amount of SDF-1 administered to the peri-infarct regionis effective to improve left ventricular end systolic volume by at leastabout 15%. In still further aspects of the application, the amount ofSDF-1 administered to the peri-infarct region is effective to improveleft ventricular end systolic volume by at least about 10%, improve leftventricular ejection fraction by at least about 10%, improve wall motionscore index by about 5%, improve six minute walk distance at least about30 meters, or improve NYHA class by at least 1 class.

In an aspect of the application, the SDF-1 plasmid can be administeredto the weakened, ischemic, and/or peri-infarct region in multipleinjections of the solution with each injection comprising about 0.33mg/ml to about 5 mg/ml of SDF-1 plasmid/solution. In one example, theSDF-1 plasmid can be administered to the weakened, ischemic, and/orperi-infarct region in at least about 10 injections. Each injectionadministered to the weakened, ischemic, and/or peri-infarct region canhave a volume of at least about 0.2 ml. The SDF-1 can be expressed inthe weakened, ischemic, and/or peri-infarct region for greater thanabout three days.

In an example application, each injection of solution comprising SDF-1expressing plasmid can have an injection volume of at least about 0.2 mland an SDF-1 plasmid concentration per injection of about 0.33 mg/ml toabout 5 mg/ml. In another aspect of the application, left ventricularend systolic volume of the of the heart can be improved can be improvedat about 10% by injecting the SDF-1 plasmid into the weakened, ischemic,and/or peri-infarct region of the heart at an injection volume per siteof at least about 0.2 ml, in at least about 10 injection sites, and atan SDF-1 plasmid concentration per injection of about 0.33 mg/ml toabout 5 mg/ml.

In a further example, the amount of SDF-1 plasmid administered to theweakened, ischemic, and/or peri-infarct region that can improve leftventricular end systolic volume is greater than about 4 mg. The volumeof solution of SDF-1 plasmid administered to the weakened, ischemic,and/or peri-infarct region that can improve left ventricular endsystolic volume of the heart is at least about 10 ml.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features of the application will become apparentto those skilled in the art to which the application relates uponreading the following description with reference to the accompanyingdrawings.

FIG. 1 is a chart illustrating luciferase expression for varying amountsand volume of DNA in a porcine model;

FIG. 2 is a chart illustrating % change of left ventricular end systolicvolume for various amounts of SDF-1 plasmid using a porcine model ofcongestive heart failure 30 days following SDF-1 injection;

FIG. 3 is a chart illustrating % change of left ventricular ejectionfraction for various amounts of SDF-1 plasmid using a porcine model ofcongestive heart failure 30 days following SDF-1 injection;

FIG. 4 is a chart illustrating % change in wall motion score index forvarious amounts of SDF-1 plasmid using a porcine model of congestiveheart failure 30 days following SDF-1 injection;

FIG. 5 is a chart illustrating % change of left ventricular end systolicvolume for various amounts of SDF-1 plasmid using a porcine model ofcongestive heart failure 90 days following SDF-1 injection; and

FIG. 6 is a chart illustrating % change of vessel density for variousamounts of SDF-1 plasmid using a porcine model of congestive heartfailure 30 days following SDF-1 injection.

FIG. 7 is a schematic diagram of a plasmid vector in accordance with anaspect of the application.

FIG. 8 is an image showing plasmid expression over a substantial portionof a porcine heart.

FIG. 9 is a chart illustrating left ventricular end systolic volume atbaseline and 30 days post-initial injection. All groups show similarincreases in left ventricular end systolic volume at 30 days. N=3 forall data points. Data presented as mean±SEM.

FIG. 10 is a chart illustrating left ventricular ejection fraction atbaseline and 30 days post-initial injection. All groups show lack ofimprovement in left ventricular ejection fraction. N=3 for all datapoints. Data presented as mean±SEM.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the application(s) belong. All patents, patentapplications, published applications and publications, Genbanksequences, websites and other published materials referred to throughoutthe entire disclosure herein, unless noted otherwise, are incorporatedby reference in their entirety. In the event that there are a pluralityof definitions for terms herein, those in this section prevail. Unlessotherwise defined, all technical terms used herein have the same meaningas commonly understood by one of ordinary skill in the art to which thisapplication belongs. Commonly understood definitions of molecularbiology terms can be found in, for example, Rieger et al., Glossary ofGenetics: Classical and Molecular, 5th edition, Springer-Verlag: NewYork, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994.

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises, such as Molecular Cloning:A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates). Methodsfor chemical synthesis of nucleic acids are discussed, for example, inBeaucage and Carruthers, Tetra. Letts. 22:1859-1862, 1981, and Matteucciet al., J. Am. Chem. Soc. 103:3185, 1981. Chemical synthesis of nucleicacids can be performed, for example, on commercial automatedoligonucleotide synthesizers. Immunological methods (e.g., preparationof antigen-specific antibodies, immunoprecipitation, and immunoblotting)are described, e.g., in Current Protocols in Immunology, ed. Coligan etal., John Wiley & Sons, New York, 1991; and Methods of ImmunologicalAnalysis, ed. Masseyeff et al., John Wiley & Sons, New York, 1992.Conventional methods of gene transfer and gene therapy can also beadapted for use in the application. See, e.g., Gene Therapy: Principlesand Applications, ed. T. Blackenstein, Springer Verlag, 1999; GeneTherapy Protocols (Methods in Molecular Medicine), ed. P. D. Robbins,Humana Press, 1997; and Retro-vectors for Human Gene Therapy, ed. C. P.Hodgson, Springer Verlag, 1996.

Where reference is made to a URL or other such identifier or address, itunderstood that such identifiers can change and particular informationon the internet can come and go, but equivalent information can be foundby searching the internet. Reference thereto evidences the availabilityand public dissemination of such information.

As used herein, “nucleic acid” refers to a polynucleotide containing atleast two covalently linked nucleotide or nucleotide analog subunits. Anucleic acid can be a deoxyribonucleic acid (DNA), a ribonucleic acid(RNA), or an analog of DNA or RNA. Nucleotide analogs are commerciallyavailable and methods of preparing polynucleotides containing suchnucleotide analogs are known (Lin et al. (1994) Nucl. Acids Res.22:5220-5234; Jellinek et al. (1995) Biochemistry 34:11363-11372;Pagratis et al. (1997) Nature Biotechnol. 15:68-73). The nucleic acidcan be single-stranded, double-stranded, or a mixture thereof. Forpurposes herein, unless specified otherwise, the nucleic acid isdouble-stranded, or it is apparent from the context.

As used herein, “DNA” is meant to include all types and sizes of DNAmolecules including cDNA, plasmids and DNA including modifiednucleotides and nucleotide analogs.

As used herein, “nucleotides” include nucleoside mono-, di-, andtriphosphates. Nucleotides also include modified nucleotides, such as,but are not limited to, phosphorothioate nucleotides and deazapurinenucleotides and other nucleotide analogs.

As used herein, the term “subject” or “patient” refers to animals intowhich the large DNA molecules can be introduced. Included are higherorganisms, such as mammals and birds, including humans, primates,rodents, cattle, pigs, rabbits, goats, sheep, mice, rats, guinea pigs,cats, dogs, horses, chicken and others.

As used herein “large mammal” refers to mammals having a typical adultweight of at least 10 kg. Such large mammals can include, for example,humans, primates, dogs, pigs, cattle and is meant to exclude smallermammals, such as mice, rats, guinea pigs, and other rodents.

As used herein, “administering to a subject” is a procedure by which oneor more delivery agents and/or large nucleic acid molecules, together orseparately, are introduced into or applied onto a subject such thattarget cells which are present in the subject are eventually contactedwith the agent and/or the large nucleic acid molecules.

As used herein, “delivery,” which is used interchangeably with“transduction,” refers to the process by which exogenous nucleic acidmolecules are transferred into a cell such that they are located insidethe cell. Delivery of nucleic acids is a distinct process fromexpression of nucleic acids.

As used herein, a “multiple cloning site (MCS)” is a nucleic acid regionin a plasmid that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector. “Restriction enzyme digestion” refers to catalyticcleavage of a nucleic acid molecule with an enzyme that functions onlyat specific locations in a nucleic acid molecule. Many of theserestriction enzymes are commercially available. Use of such enzymes iswidely understood by those of skill in the art. Frequently, a vector islinearized or fragmented using a restriction enzyme that cuts within theMCS to enable exogenous sequences to be ligated to the vector.

As used herein, “origin of replication” (often termed “ori”), is aspecific nucleic acid sequence at which replication is initiated.Alternatively, an autonomously replicating sequence (ARS) can beemployed if the host cell is yeast.

As used herein, “selectable or screenable markers” confer anidentifiable change to a cell permitting easy identification of cellscontaining an expression vector. Generally, a selectable marker is onethat confers a property that allows for selection. A positive selectablemarker is one in which the presence of the marker allows for itsselection, while a negative selectable marker is one in which itspresence prevents its selection. An example of a positive selectablemarker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscalorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

The term “transfection” is used to refer to the uptake of foreign DNA bya cell. A cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare generally known in the art. See, e.g., Graham et al., Virology52:456 (1973); Sambrook et al., Molecular Cloning: A Laboratory Manual(1989); Davis et al., Basic Methods in Molecular Biology (1986); Chu etal., Gene 13:197 (1981). Such techniques can be used to introduce one ormore exogenous DNA moieties, such as a nucleotide integration vector andother nucleic acid molecules, into suitable host cells. The termcaptures chemical, electrical, and viral-mediated transfectionprocedures.

As used herein, “expression” refers to the process by which nucleic acidis translated into peptides or is transcribed into RNA, which, forexample, can be translated into peptides, polypeptides or proteins. Ifthe nucleic acid is derived from genomic DNA, expression may, if anappropriate eukaryotic host cell or organism is selected, includesplicing of the mRNA. For heterologous nucleic acid to be expressed in ahost cell, it must initially be delivered into the cell and then, oncein the cell, ultimately reside in the nucleus.

As used herein, “genetic therapy” involves the transfer of heterologousDNA to cells of a mammal, particularly a human, with a disorder orconditions for which therapy or diagnosis is sought. The DNA isintroduced into the selected target cells in a manner such that theheterologous DNA is expressed and a therapeutic product encoded therebyis produced. Alternatively, the heterologous DNA may in some mannermediate expression of DNA that encodes the therapeutic product; it mayencode a product, such as a peptide or RNA that in some manner mediates,directly or indirectly, expression of a therapeutic product. Genetictherapy may also be used to deliver nucleic acid encoding a gene productto replace a defective gene or supplement a gene product produced by themammal or the cell in which it is introduced. The introduced nucleicacid may encode a therapeutic compound, such as a growth factorinhibitor thereof, or a tumor necrosis factor or inhibitor thereof, suchas a receptor therefore, that is not normally produced in the mammalianhost or that is not produced in therapeutically effective amounts or ata therapeutically useful time. The heterologous DNA encoding thetherapeutic product may be modified prior to introduction into the cellsof the afflicted host in order to enhance or otherwise alter the productor expression thereof.

As used herein, “heterologous nucleic acid sequence” is typically DNAthat encodes RNA and proteins that are not normally produced in vivo bythe cell in which it is expressed or that mediates or encodes mediatorsthat alter expression of endogenous DNA by affecting transcription,translation, or other regulatable biochemical processes. A heterologousnucleic acid sequence may also be referred to as foreign DNA. Any DNAthat one of skill in the art would recognize or consider as heterologousor foreign to the cell in which it is expressed is herein encompassed byheterologous DNA. Examples of heterologous DNA include, but are notlimited to, DNA that encodes traceable marker proteins, such as aprotein that confers drug resistance, DNA that encodes therapeuticallyeffective substances, such as anti-cancer agents, enzymes and hormones,and DNA that encodes other types of proteins, such as antibodies.Antibodies that are encoded by heterologous DNA may be secreted orexpressed on the surface of the cell in which the heterologous DNA hasbeen introduced.

As used herein the term “cardiomyopathy” refers to the deterioration ofthe function of the myocardium (i.e., the actual heart muscle) for anyreason. Subjects with cardiomyopathy are often at risk of arrhythmia,sudden cardiac death, or hospitalization or death due to heart failure.

As used herein, the term “ischemic cardiomyopathy” is a weakness in themuscle of the heart due to inadequate oxygen delivery to the myocardiumwith coronary artery disease being the most common cause.

As used herein the term “ischemic cardiac disease” refers to anycondition in which heart muscle is damaged or works inefficientlybecause of an absence or relative deficiency of its blood supply; mostoften caused by atherosclerosis, it includes angina pectoris, acutemyocardial infarction, chronic ischemic heart disease, and sudden death.

As used herein the term “myocardial infarction” refers to the damagingor death of an area of the heart muscle (myocardium) resulting from ablocked blood supply to that area.

As used herein the term “6-minute walk test” or “6MWT” refers to a testthat measures the distance that a patient can quickly walk on a flat,hard surface in a period of 6 minutes (the 6MWD). It evaluates theglobal and integrated responses of all the systems involved duringexercise, including the pulmonary and cardiovascular systems, systemiccirculation, peripheral circulation, blood, neuromuscular units, andmuscle metabolism. It does not provide specific information on thefunction of each of the different organs and systems involved inexercise or the mechanism of exercise limitation, as is possible withmaximal cardiopulmonary exercise testing. The self-paced 6MWT assessesthe submaximal level of functional capacity. (See for example, AM JRespir Crit Care Med, Vol. 166. Pp 111-117 (2002))

As used herein “New York Heart Association (NYHA) functionalclassification” refers to a classification for the extent of heartfailure. It places patients in one of four categories based on how muchthey are limited during physical activity; the limitations/symptoms arein regards to normal breathing and varying degrees in shortness ofbreath and or angina pain:

NYHA Class Symptoms I No symptoms and no limitation in ordinary physicalactivity, e.g. shortness of breath when walking, climbing stairs etc. IIMild symptoms (mild shortness of breath and/or angina) and slightlimitation during ordinary activity. III Marked limitation in activitydue to symptoms, even during less-than-ordinary activity, e.g. walkingshort distances (20-100 m). Comfortable only at rest. IV Severelimitations. Experiences symptoms even while at rest. Mostly bedboundpatients.

This application relates to compositions and methods of treating acardiomyopathy in a subject that results in reduced and/or impairedmyocardial function. The cardiomyopathy treated by the compositions andmethods herein can include cardiomyopathies associated with a pulmonaryembolus, a venous thrombosis, a myocardial infarction, a transientischemic attack, a peripheral vascular disorder, atherosclerosis,ischemic cardiac disease and/or other myocardial injury or vasculardisease. The method of treating the cardiomyopathy can include locallyadministering (or locally delivering) to weakened myocardial tissue,ischemic myocardial tissue, and/or apoptotic myocardial tissue, such asthe peri-infarct region of a heart following myocardial infarction, anamount of stromal-cell derived factor-1 (SDF-1) that is effective tocause functional improvement in at least one of the followingparameters: left ventricular volume, left ventricular area, leftventricular dimension, cardiac function, 6-minute walk test (6MWT), orNew York Heart Association (NYHA) functional classification.

It was found using a porcine model of heart failure that mimics heartfailure in a human that functional improvement of ischemic myocardialtissue is dependent on the amount, dose, and/or delivery of SDF-1administered to the ischemic myocardial tissue and that the amount,dose, and/or delivery of SDF-1 to the ischemic myocardial tissue can beoptimized so that myocardial functional parameters, such as leftventricular volume, left ventricular area, left ventricular dimension,or cardiac function are substantially improved. As discussed below, insome aspects, the amount, concentration, and volume of SDF-1administered to the ischemic myocardial tissue can be controlled and/oroptimized to substantially improve the functional parameters (e.g., leftventricular volume, left ventricular area, left ventricular dimension,cardiac function, 6-minute walk test (6MWT), and/or New York HeartAssociation (NYHA) functional classification) while mitigating adverseside effects.

In one example, the SDF-1 can be administered directly or locally to aweakened region, an ischemic region, and/or peri-infarct region ofmyocardial tissue of a large mammal (e.g., pig or human) in which thereis a deterioration or worsening of a functional parameter of the heart,such as left ventricular volume, left ventricular area, left ventriculardimension, or cardiac function as a result of an ischemiccardiomyopathy, such as a myocardial infarction. The deterioration orworsening of the functional parameter can include, for example, anincrease in left ventricular end systolic volume, decrease in leftventricular ejection fraction, increase in wall motion score index,increase in left ventricular end diastolic length, increase in leftventricular end systolic length, increase in left ventricular enddiastolic area (e.g., mitral valve level and papillary muscle insertionlevel), increase in left ventricular end systolic area (e.g., mitralvalve level and papillary muscle insertion level), or increase in leftventricular end diastolic volume as measured using, for example, usingechocardiography.

In an aspect of the application, the amount of SDF-1 administered to theweakened region, ischemic region, and/or peri-infarct region of themyocardial tissue of the large mammal can be an amount effective toimprove at least one functional parameter of the myocardium, such as adecrease in left ventricular end systolic volume, increase in leftventricular ejection fraction, decrease in wall motion score index,decrease in left ventricular end diastolic length, decrease in leftventricular end systolic length, decrease in left ventricular enddiastolic area (e.g., mitral valve level and papillary muscle insertionlevel), decrease in left ventricular end systolic area (e.g., mitralvalve level and papillary muscle insertion level), or decrease in leftventricular end diastolic volume measured using, for example, usingechocardiography as well as improve the subject's 6-minute walk test(6MWT) or New York Heart Association (NYHA) functional classification.

In another aspect of the application, the amount of SDF-1 administeredto the weakened region, ischemic region, and/or peri-infarct region ofthe myocardial tissue of the large mammal with a cardiomyopathy iseffective to improve left ventricular end systolic volume in the mammalby at least about 10%, and more specifically at least about 15%, after30 days following administration as measured by echocardiography. Thepercent improvement is relative to each subject treated and is based onthe respective parameter measured prior to or at the time of therapeuticintervention or treatment.

In a further aspect of the application, the amount of SDF-1 administeredto the weakened region, ischemic region, and/or peri-infarct region ofthe myocardial tissue of the large mammal with a cardiomyopathy iseffective to improve left ventricular end systolic volume by at leastabout 10%, improve left ventricular ejection fraction by at least about10%, and improve wall motion score index by about 5%, after 30 daysfollowing administration as measured by echocardiography.

In a still further aspect of the application, the amount of SDF-1administered to the weakened region, ischemic region, and/orperi-infarct region of the myocardial tissue of the large mammal with acardiomyopathy is effective to improve vasculogenesis of the weakenedregion, ischemic region, and/or peri-infarct region by at least 20%based on vessel density or an increase in cardiac perfusion measured bySPECT imaging. A 20% improvement in vasculogenesis has been shown to beclinically significant (Losordo Circulation 2002; 105:2012).

In a still further aspect of the application, the amount of SDF-1administered to the weakened region, ischemic region, and/orperi-infarct region of the myocardial tissue of the large mammal with acardiomyopathy is effective to improve six minute walk distance at leastabout 30 meters or improve NYHA class by at least 1 class.

The SDF-1 described herein can be administered to the weakened region,the ischemic region, and/or peri-infarct region of the myocardial tissuefollowing tissue injury (e.g., myocardial infarction) to about hours,days, weeks, or months after onset of down-regulation of SDF-1. Theperiod of time that the SDF-1 is administered to the cells can comprisefrom about immediately after onset of the cardiomyopathy (e.g.,myocardial infarction) to about days, weeks, or months after the onsetof the ischemic disorder or tissue injury.

SDF-1 in accordance with the application that is administered to theweakened, ischemic, and/or a peri-infarct region of the myocardialtissue peri-infarct region can have an amino acid sequence that issubstantially similar to a native mammalian SDF-1 amino acid sequence.The amino acid sequence of a number of different mammalian SDF-1 proteinare known including human, mouse, and rat. The human and rat SDF-1 aminoacid sequences are at least about 92% identical (e.g., about 97%identical). SDF-1 can comprise two isoforms, SDF-1 alpha and SDF-1 beta,both of which are referred to herein as SDF-1 unless identifiedotherwise.

The SDF-1 can have an amino acid sequence substantially identical to SEQID NO: 1. The SDF-1 that is over-expressed can also have an amino acidsequence substantially similar to one of the foregoing mammalian SDF-1proteins. For example, the SDF-1 that is over-expressed can have anamino acid sequence substantially similar to SEQ ID NO: 2. SEQ ID NO: 2,which substantially comprises SEQ ID NO: 1, is the amino acid sequencefor human SDF-1 and is identified by GenBank Accession No. NP954637. TheSDF-1 that is over-expressed can also have an amino acid sequence thatis substantially identical to SEQ ID NO: 3. SEQ ID NO: 3 includes theamino acid sequences for rat SDF and is identified by GenBank AccessionNo. AAF01066.

The SDF-1 in accordance with the application can also be a variant ofmammalian SDF-1, such as a fragment, analog and derivative of mammalianSDF-1. Such variants include, for example, a polypeptide encoded by anaturally occurring allelic variant of native SDF-1 gene (i.e., anaturally occurring nucleic acid that encodes a naturally occurringmammalian SDF-1 polypeptide), a polypeptide encoded by an alternativesplice form of a native SDF-1 gene, a polypeptide encoded by a homologor ortholog of a native SDF-1 gene, and a polypeptide encoded by anon-naturally occurring variant of a native SDF-1 gene.

SDF-1 variants have a peptide sequence that differs from a native SDF-1polypeptide in one or more amino acids. The peptide sequence of suchvariants can feature a deletion, addition, or substitution of one ormore amino acids of a SDF-1 variant. Amino acid insertions arepreferably of about 1 to 4 contiguous amino acids, and deletions arepreferably of about 1 to 10 contiguous amino acids. Variant SDF-1polypeptides substantially maintain a native SDF-1 functional activity.Examples of SDF-1 polypeptide variants can be made by expressing nucleicacid molecules that feature silent or conservative changes. One exampleof an SDF-1 variant is listed in U.S. Pat. No. 7,405,195, which isherein incorporated by reference in its entirety.

SDF-1 polypeptide fragments corresponding to one or more particularmotifs and/or domains or to arbitrary sizes, are within the scope ofthis application. Isolated peptidyl portions of SDF-1 can be obtained byscreening peptides recombinantly produced from the correspondingfragment of the nucleic acid encoding such peptides. For example, anSDF-1 polypeptide may be arbitrarily divided into fragments of desiredlength with no overlap of the fragments, or preferably divided intooverlapping fragments of a desired length. The fragments can be producedrecombinantly and tested to identify those peptidyl fragments, which canfunction as agonists of native CXCR-4 polypeptides.

Variants of SDF-1 polypeptides can also include recombinant forms of theSDF-1 polypeptides. Recombinant polypeptides in some embodiments, inaddition to SDF-1 polypeptides, are encoded by a nucleic acid that canhave at least 70% sequence identity with the nucleic acid sequence of agene encoding a mammalian SDF-1.

SDF-1 variants can include agonistic forms of the protein thatconstitutively express the functional activities of native SDF-1. OtherSDF-1 variants can include those that are resistant to proteolyticcleavage, as for example, due to mutations, which alter protease targetsequences. Whether a change in the amino acid sequence of a peptideresults in a variant having one or more functional activities of anative SDF-1 can be readily determined by testing the variant for anative SDF-1 functional activity.

The SDF-1 nucleic acid that encodes the SDF-1 protein can be a native ornon-native nucleic acid and be in the form of RNA or in the form of DNA(e.g., cDNA, genomic DNA, and synthetic DNA). The DNA can bedouble-stranded or single-stranded, and if single-stranded may be thecoding (sense) strand or non-coding (anti-sense) strand. The nucleicacid coding sequence that encodes SDF-1 may be substantially similar toa nucleotide sequence of the SDF-1 gene, such as nucleotide sequenceshown in SEQ ID NO: 4 and SEQ ID NO: 5. SEQ ID NO: 4 and SEQ ID NO: 5comprise, respectively, the nucleic acid sequences for human SDF-1 andrat SDF-1 and are substantially similar to the nucleic sequences ofGenBank Accession No. NM199168 and GenBank Accession No. AF189724. Thenucleic acid coding sequence for SDF-1 can also be a different codingsequence which, as a result of the redundancy or degeneracy of thegenetic code, encodes the same polypeptide as SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO: 3.

Other nucleic acid molecules that encode SDF-1 are variants of a nativeSDF-1, such as those that encode fragments, analogs and derivatives ofnative SDF-1. Such variants may be, for example, a naturally occurringallelic variant of a native SDF-1 gene, a homolog or ortholog of anative SDF-1 gene, or a non-naturally occurring variant of a nativeSDF-1 gene. These variants have a nucleotide sequence that differs froma native SDF-1 gene in one or more bases. For example, the nucleotidesequence of such variants can feature a deletion, addition, orsubstitution of one or more nucleotides of a native SDF-1 gene. Nucleicacid insertions are preferably of about 1 to 10 contiguous nucleotides,and deletions are preferably of about 1 to 10 contiguous nucleotides.

In other applications, variant SDF-1 displaying substantial changes instructure can be generated by making nucleotide substitutions that causeless than conservative changes in the encoded polypeptide. Examples ofsuch nucleotide substitutions are those that cause changes in (a) thestructure of the polypeptide backbone; (b) the charge or hydrophobicityof the polypeptide; or (c) the bulk of an amino acid side chain.Nucleotide substitutions generally expected to produce the greatestchanges in protein properties are those that cause non-conservativechanges in codons. Examples of codon changes that are likely to causemajor changes in protein structure are those that cause substitution of(a) a hydrophilic residue(e.g., serine or threonine), for (or by) ahydrophobic residue (e.g., leucine, isoleucine, phenylalanine, valine oralanine); (b) a cysteine or proline for (or by) any other residue; (c) aresidue having an electropositive side chain (e.g., lysine, arginine, orhistidine), for (or by) an electronegative residue (e.g., glutamine oraspartine); or (d) a residue having a bulky side chain (e.g.,phenylalanine), for (or by) one not having a side chain, (e.g.,glycine).

Naturally occurring allelic variants of a native SDF-1 gene are nucleicacids isolated from mammalian tissue that have at least 70% sequenceidentity with a native SDF-1 gene, and encode polypeptides havingstructural similarity to a native SDF-1 polypeptide. Homologs of anative SDF-1 gene are nucleic acids isolated from other species thathave at least 70% sequence identity with the native gene, and encodepolypeptides having structural similarity to a native SDF-1 polypeptide.Public and/or proprietary nucleic acid databases can be searched toidentify other nucleic acid molecules having a high percent (e.g., 70%or more) sequence identity to a native SDF-1 gene.

Non-naturally occurring SDF-1 gene variants are nucleic acids that donot occur in nature (e.g., are made by the hand of man), have at least70% sequence identity with a native SDF-1 gene, and encode polypeptideshaving structural similarity to a native SDF-1 polypeptide. Examples ofnon-naturally occurring SDF-1 gene variants are those that encode afragment of a native SDF-1 protein, those that hybridize to a nativeSDF-1 gene or a complement of to a native SDF-1 gene under stringentconditions, and those that share at least 65% sequence identity with anative SDF-1 gene or a complement of a native SDF-1 gene.

Nucleic acids encoding fragments of a native SDF-1 gene in someembodiments are those that encode amino acid residues of native SDF-1.Shorter oligonucleotides that encode or hybridize with nucleic acidsthat encode fragments of native SDF-1 can be used as probes, primers, orantisense molecules. Longer polynucleotides that encode or hybridizewith nucleic acids that encode fragments of a native SDF-1 can also beused in various aspects of the application. Nucleic acids encodingfragments of a native SDF-1 can be made by enzymatic digestion (e.g.,using a restriction enzyme) or chemical degradation of the full-lengthnative SDF-1 gene or variants thereof.

Nucleic acids that hybridize under stringent conditions to one of theforegoing nucleic acids can also be used herein. For example, suchnucleic acids can be those that hybridize to one of the foregoingnucleic acids under low stringency conditions, moderate stringencyconditions, or high stringency conditions.

Nucleic acid molecules encoding a SDF-1 fusion protein may also be usedin some embodiments. Such nucleic acids can be made by preparing aconstruct (e.g., an expression vector) that expresses a SDF-1 fusionprotein when introduced into a suitable target cell. For example, such aconstruct can be made by ligating a first polynucleotide encoding aSDF-1 protein fused in frame with a second polynucleotide encodinganother protein such that expression of the construct in a suitableexpression system yields a fusion protein.

The nucleic acids encoding SDF-1 can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The nucleic acids described hereinmay additionally include other appended groups such as peptides (e.g.,for targeting target cell receptors in vivo), or agents facilitatingtransport across the cell membrane, hybridization-triggered cleavage. Tothis end, the nucleic acids may be conjugated to another molecule,(e.g., a peptide), hybridization triggered cross-linking agent,transport agent, hybridization-triggered cleavage agent, etc.

The SDF-1 can be delivered to the weakened, ischemic, and/orperi-infarct region of the myocardial tissue by administering an SDF-1protein to the to the weakened, ischemic, and/or peri-infarct region, orby introducing an agent into cells of the weakened region, ischemicregion, and/or peri-infarct region of the myocardial tissue that causes,increases, and/or upregulates expression of SDF-1 (i.e., SDF-1 agent).The SDF-1 protein expressed from the cells can be an expression productof a genetically modified cell.

The agent that causes, increases, and/or upregulates expression of SDF-1can comprise natural or synthetic nucleic acids as described herein thatare incorporated into recombinant nucleic acid constructs, typically DNAconstructs, capable of introduction into and replication in the cells ofthe myocardial tissue. Such a construct can include a replication systemand sequences that are capable of transcription and translation of apolypeptide-encoding sequence in a given cell.

One method of introducing the agent into a target cell involves usinggene therapy. Gene therapy in some embodiments of the application can beused to express SDF-1 protein from a cell of the weakened region,ischemic region, and/or peri-infarct region of the myocardial tissue invivo.

In an aspect of the application, the gene therapy can use a vectorincluding a nucleotide encoding an SDF-1 protein. A “vector” (sometimesreferred to as gene delivery or gene transfer “vehicle”) refers to amacromolecule or complex of molecules comprising a polynucleotide to bedelivered to a target cell, either in vitro or in vivo. Thepolynucleotide to be delivered may comprise a coding sequence ofinterest in gene therapy. Vectors include, for example, viral vectors(such as adenoviruses ('Ad'), adeno-associated viruses (AAV), andretroviruses), non-viral vectors, liposomes, and other lipid-containingcomplexes, and other macromolecular complexes capable of mediatingdelivery of a polynucleotide to a target cell.

Vectors can also comprise other components or functionalities thatfurther modulate gene delivery and/or gene expression, or that otherwiseprovide beneficial properties to the targeted cells. Such othercomponents include, for example, components that influence binding ortargeting to cells (including components that mediate cell-type ortissue-specific binding); components that influence uptake of the vectornucleic acid by the cell; components that influence localization of thepolynucleotide within the cell after uptake (such as agents mediatingnuclear localization); and components that influence expression of thepolynucleotide. Such components also might include markers, such asdetectable and/or selectable markers that can be used to detect orselect for cells that have taken up and are expressing the nucleic aciddelivered by the vector. Such components can be provided as a naturalfeature of the vector (such as the use of certain viral vectors whichhave components or functionalities mediating binding and uptake), orvectors can be modified to provide such functionalities.

Selectable markers can be positive, negative or bifunctional. Positiveselectable markers allow selection for cells carrying the marker,whereas negative selectable markers allow cells carrying the marker tobe selectively eliminated. A variety of such marker genes have beendescribed, including bifunctional (i.e., positive/negative) markers(see, e.g., Lupton, S., WO 92/08796, published May 29, 1992; and Lupton,S., WO 94/28143, published Dec. 8, 1994). Such marker genes can providean added measure of control that can be advantageous in gene therapycontexts. A large variety of such vectors are known in the art and aregenerally available.

Vectors for use herein include viral vectors, lipid based vectors andother non-viral vectors that are capable of delivering a nucleotide tothe cells of weakened region, ischemic region, and/or peri-infarctregion of the myocardial tissue. The vector can be a targeted vector,especially a targeted vector that preferentially binds to the cells ofweakened region, ischemic region, and/or peri-infarct region of themyocardial tissue. Viral vectors for use in the methods herein caninclude those that exhibit low toxicity to the cells of weakened region,ischemic region, and/or peri-infarct region of the myocardial tissue andinduce production of therapeutically useful quantities of SDF-1 proteinin a tissue-specific manner.

Examples of viral vectors are those derived from adenovirus (Ad) oradeno-associated virus (AAV). Both human and non-human viral vectors canbe used and the recombinant viral vector can be replication-defective inhumans. Where the vector is an adenovirus, the vector can comprise apolynucleotide having a promoter operably linked to a gene encoding theSDF-1 protein and is replication-defective in humans.

Other viral vectors that can be use in accordance with method of theapplication include herpes simplex virus (HSV)-based vectors. HSVvectors deleted of one or more immediate early genes (IE) areadvantageous because they are generally non-cytotoxic, persist in astate similar to latency in the target cell, and afford efficient targetcell transduction. Recombinant HSV vectors can incorporate approximately30 kb of heterologous nucleic acid.

Retroviruses, such as C-type retroviruses and lentiviruses, might alsobe used in some embodiments of the application. For example, retroviralvectors may be based on murine leukemia virus (MLV). See, e.g., Hu andPathak, Pharmacol. Rev. 52:493-511, 2000 and Fong et al., Crit. Rev.Ther. Drug Carrier Syst. 17:1-60, 2000. MLV-based vectors may contain upto 8 kb of heterologous (therapeutic) DNA in place of the viral genes.The heterologous DNA may include a tissue-specific promoter and an SDF-1nucleic acid. In methods of delivery to cells proximate the wound, itmay also encode a ligand to a tissue specific receptor.

Additional retroviral vectors that might be used arereplication-defective lentivirus-based vectors, including humanimmunodeficiency (HIV)-based vectors. See, e.g., Vigna and Naldini, J.Gene Med. 5:308-316, 2000 and Miyoshi et al., J. Virol. 72:8150-8157,1998. Lentiviral vectors are advantageous in that they are capable ofinfecting both actively dividing and non-dividing cells. They are alsohighly efficient at transducing human epithelial cells.

Lentiviral vectors for use in the methods herein may be derived fromhuman and non-human (including SIV) lentiviruses. Examples of lentiviralvectors include nucleic acid sequences required for vector propagationas well as a tissue-specific promoter operably linked to a SDF-1 gene.These former may include the viral LTRs, a primer binding site, apolypurine tract, att sites, and an encapsidation site.

A lentiviral vector may be packaged into any suitable lentiviral capsid.The substitution of one particle protein with another from a differentvirus is referred to as “pseudotyping”. The vector capsid may containviral envelope proteins from other viruses, including murine leukemiavirus (MLV) or vesicular stomatitis virus (VSV). The use of the VSVG-protein yields a high vector titer and results in greater stability ofthe vector virus particles.

Alphavirus-based vectors, such as those made from semliki forest virus(SFV) and sindbis virus (SIN) might also be used herein. Use ofalphaviruses is described in Lundstrom, K., Intervirology 43:247-257,2000 and Perri et al., Journal of Virology 74:9802-9807, 2000.

Recombinant, replication-defective alphavirus vectors are advantageousbecause they are capable of high-level heterologous (therapeutic) geneexpression, and can infect a wide target cell range. Alphavirusreplicons may be targeted to specific cell types by displaying on theirvirion surface a functional heterologous ligand or binding domain thatwould allow selective binding to target cells expressing a cognatebinding partner. Alphavirus replicons may establish latency, andtherefore long-term heterologous nucleic acid expression in a targetcell. The replicons may also exhibit transient heterologous nucleic acidexpression in the target cell.

In many of the viral vectors compatible with methods of the application,more than one promoter can be included in the vector to allow more thanone heterologous gene to be expressed by the vector. Further, the vectorcan comprise a sequence which encodes a signal peptide or other moietywhich facilitates the expression of a SDF-1 gene product from the targetcell.

To combine advantageous properties of two viral vector systems, hybridviral vectors may be used to deliver a SDF-1 nucleic acid to a targettissue. Standard techniques for the construction of hybrid vectors arewell-known to those skilled in the art. Such techniques can be found,for example, in Sambrook, et al., In Molecular Cloning: A laboratorymanual. Cold Spring Harbor, N.Y. or any number of laboratory manualsthat discuss recombinant DNA technology. Double-stranded AAV genomes inadenoviral capsids containing a combination of AAV and adenoviral ITRsmay be used to transduce cells. In another variation, an AAV vector maybe placed into a “gutless”, “helper-dependent” or “high-capacity”adenoviral vector. Adenovirus/AAV hybrid vectors are discussed in Lieberet al., J. Virol. 73:9314-9324, 1999. Retrovirus/adenovirus hybridvectors are discussed in Zheng et al., Nature Biotechnol. 18:176-186,2000. Retroviral genomes contained within an adenovirus may integratewithin the target cell genome and effect stable SDF-1 gene expression.

Other nucleotide sequence elements which facilitate expression of theSDF-1 gene and cloning of the vector are further contemplated. Forexample, the presence of enhancers upstream of the promoter orterminators downstream of the coding region, for example, can facilitateexpression.

In accordance with another aspect of the application, a tissue-specificpromoter, can be fused to a SDF-1 gene. By fusing such tissue specificpromoter within the adenoviral construct, transgene expression islimited to a particular tissue. The efficacy of gene expression anddegree of specificity provided by tissue specific promoters can bedetermined, using the recombinant adenoviral system described herein.

In addition to viral vector-based methods, non-viral methods may also beused to introduce a SDF-1 nucleic acid into a target cell. A review ofnon-viral methods of gene delivery is provided in Nishikawa and Huang,Human Gene Ther. 12:861-870, 2001. An example of a non-viral genedelivery method according to the invention employs plasmid DNA tointroduce a SDF-1 nucleic acid into a cell. Plasmid-based gene deliverymethods are generally known in the art. In one example, the plasmidvector can have a structure as shown schematically in FIG. 7. Theplasmid vector of FIG. 7 includes a CMV enhancer and CMV promoterupstream of an SDF-1α cDNA (RNA) sequence.

Optionally, a synthetic gene transfer molecules can be designed to formmultimolecular aggregates with plasmid SDF-1 DNA. These aggregates canbe designed to bind to cells of weakened region, ischemic region, and/orperi-infarct region of the myocardial tissue. Cationic amphiphiles,including lipopolyamines and cationic lipids, may be used to providereceptor-independent SDF-1 nucleic acid transfer into target cells(e.g., cardiomyocytes). In addition, preformed cationic liposomes orcationic lipids may be mixed with plasmid DNA to generatecell-transfecting complexes. Methods involving cationic lipidformulations are reviewed in Felgner et al., Ann. N.Y. Acad. Sci.772:126-139, 1995 and Lasic and Templeton, Adv. Drug Delivery Rev.20:221-266, 1996. For gene delivery, DNA may also be coupled to anamphipathic cationic peptide (Fominaya et al., J. Gene Med. 2:455-464,2000).

Methods that involve both viral and non-viral based components may beused herein. For example, an Epstein Barr virus (EBV)-based plasmid fortherapeutic gene delivery is described in Cui et al., Gene Therapy8:1508-1513, 2001. Additionally, a method involving aDNA/ligand/polycationic adjunct coupled to an adenovirus is described inCuriel, D. T., Nat. Immun. 13:141-164, 1994.

Additionally, the SDF-1 nucleic acid can be introduced into the targetcell by transfecting the target cells using electroporation techniques.Electroporation techniques are well known and can be used to facilitatetransfection of cells using plasmid DNA.

Vectors that encode the expression of SDF-1 can be delivered to thetarget cell in the form of an injectable preparation containingpharmaceutically acceptable carrier, such as saline, as necessary. Otherpharmaceutical carriers, formulations and dosages can also be used inaccordance with the present invention.

In one aspect of the invention, the vector can comprise an SDF-1plasmid, such as for example in FIG. 7. SDF-1 plasmid can be deliveredto cells of the weakened region, ischemic region, and/or peri-infarctregion of the myocardial tissue by direct injection of the SDF-1 plasmidvector into the weakened region, ischemic region, and/or peri-infarctregion of the myocardial tissue at an amount effective to improve atleast one myocardial functional parameters, such as left ventricularvolume, left ventricular area, left ventricular dimension, or cardiacfunction as well as improve the subject's 6-minute walk test (6MWT) orNew York Heart Association (NYHA) functional classification. Byinjecting the vector directly into or about the periphery of theweakened region, ischemic region, and/or peri-infarct region of themyocardial tissue, it is possible to target the vector transfectionrather effectively, and to minimize loss of the recombinant vectors.This type of injection enables local transfection of a desired number ofcells, especially about the weakened region, ischemic region, and/orperi-infarct region of the myocardial tissue, thereby maximizingtherapeutic efficacy of gene transfer, and minimizing the possibility ofan inflammatory response to viral proteins.

In an aspect of the application, the SDF-1 plasmid can be administeredto the weakened, ischemic, and/or peri-infarct region in multipleinjections of a solution of SDF-1 expressing plasmid DNA with eachinjection comprising about 0.33 mg/ml to about 5 mg/ml of SDF-1plasmid/solution. In one example, the SDF-1 plasmid can be administeredto the weakened, ischemic, and/or peri-infarct region in at least about10 injections, at least about 15 injections, or at least about 20injections. Multiple injections of the SDF-1 plasmid to the weakened,ischemic, and/or peri-infarct region allows a greater area and/or numberof cells of the weakened, ischemic, and/or peri-infarct region to betreated.

Each injection administered to the weakened, ischemic, and/orperi-infarct region can have a volume of at least about 0.2 ml. Thetotal volume of solution that includes the amount of SDF-1 plasmidadministered to the weakened, ischemic, and/or peri-infarct region thatcan improve at least one functional parameter of the heart is at leastabout 10 ml.

In one example, the SDF-1 plasmid can be administered to the weakened,ischemic, and/or peri-infarct region in at least about 10 injections.Each injection administered to the weakened, ischemic, and/orperi-infarct region can have a volume of at least about 0.2 ml. TheSDF-1 can be expressed in the weakened, ischemic, and/or peri-infarctregion for greater than about three days.

For example, each injection of solution including SDF-1 expressingplasmid can have an injection volume of at least about 0.2 ml and anSDF-1 plasmid concentration per injection of about 0.33 mg/ml to about 5mg/ml. In another aspect of the application, at least one functionalparameter of the of the heart can be improved by injecting the SDF-1plasmid into the weakened, ischemic, and/or peri-infarct region of theheart at an injection volume per site of at least about 0.2 ml, in atleast about 10 injection sites, and at an SDF-1 plasmid concentrationper injection of about 0.33 mg/ml to about 5 mg/ml.

It was found in a porcine model of congestive heart failure thatinjections of a solution of SDF-1 plasmid having concentration of lessabout 0.33 mg/ml or greater than about 5 mg/ml and an injection volumeper injection site less than about 0.2 ml to a porcine model of heartfailure resulted in little if any functional improvement of the leftventricular volume, left ventricular area, left ventricular dimension,or cardiac function of the treated heart.

In another aspect of the application, the amount of SDF-1 plasmidadministered to the weakened, ischemic, and/or peri-infarct region thatcan improve at least one functional parameter of the heart is greaterthan about 4 mg and less than about 100 mg per therapeutic intervention.The amount of SDF-1 plasmid administered by therapeutic interventionherein refers to the total SDF-1 plasmid administered to the subjectduring a therapeutic procedure designed to affect or elicit atherapeutic effect. This can include the total SDF-1 plasmidadministered in single injection for a particular therapeuticintervention or the total SDF-1 plasmid that is administered by multipleinjections for a therapeutic intervention. It was found in a porcinemodel of congestive heart failure that administration of about 4 mgSDF-1 plasmid DNA via direct injection of the SDF-1 plasmid to the heartresulted in no functional improvement of the left ventricular volume,left ventricular area, left ventricular dimension, or cardiac functionof the treated heart. Moreover, administration of about 100 mg of SDF-1plasmid DNA via direct injection of the SDF-1 plasmid to the heartresulted in no functional improvement of the left ventricular volume,left ventricular area, left ventricular dimension, or cardiac functionof the treated heart.

In some aspects of the application, the SDF-1 can be expressed at atherapeutically effective amount or dose in the weakened, ischemic,and/or peri-infarct region after transfection with the SDF-1 plasmidvector for greater than about three days. Expression of SDF-1 at atherapeutically effective dose or amount for greater three days canprovide a therapeutic effect to weakened, ischemic, and/or peri-infarctregion. Advantageously, the SDF-1 can be expressed in the weakened,ischemic, and/or peri-infarct region after transfection with the SDF-1plasmid vector at a therapeutically effective amount for less than about90 days to mitigate potentially chronic and/or cytotoxic effects thatmay inhibit the therapeutic efficacy of the administration of the SDF-1to the subject.

It will be appreciated that the amount, volume, concentration, and/ordosage of SDF-1 plasmid that is administered to any one animal or humandepends on many factors, including the subject's size, body surfacearea, age, the particular composition to be administered, sex, time androute of administration, general health, and other drugs beingadministered concurrently. Specific variations of the above notedamounts, volumes, concentrations, and/or dosages of SDF-1 plasmid can bereadily be determined by one skilled in the art using the experimentalmethods described below.

In another aspect of the application, the SDF-1 plasmid can beadministered by direct injection using catheterization, such asendo-ventricular catheterization or intra-myocardial catheterization. Inone example, a deflectable guide catheter device can be advanced to aleft ventricle retrograde across the aortic valve. Once the device ispositioned in the left ventricle, SDF-1 plasmid can be injected into theperi-infarct region (both septal and lateral aspect) area of the leftventricle. Typically, 1.0 ml of SDF-1 plasmid solution can be injectionover a period of time of about 60 seconds. The subject be treated canreceive at least about 10 injection (e.g., about 15 to about 20injections in total).

The myocardial tissue of the subject can be imaged prior toadministration of the SDF-1 plasmid to define the area of weakened,ischemic, and/or peri-infarct region prior to administration of theSDF-1 plasmid. Defining the weakened, ischemic, and/or peri-infarctregion by imaging allows for more accurate intervention and targeting ofthe SDF-1 plasmid to the weakened, ischemic, and/or peri-infarct region.The imaging technique used to define the weakened, ischemic, and/orperi-infarct region of the myocardial tissue can include any knowncardio-imaging technique. Such imaging techniques can include, forexample, at least one of echocardiography, magnetic resonance imaging,coronary angiogram, electroanatomical mapping, or fluoroscopy. It willbe appreciated that other imaging techniques that can define theweakened, ischemic, and/or peri-infarct region can also be used.

Optionally, other agents besides SDF-1 nucleic acids (e.g., SDF-1plasmids) can be introduced into the weakened, ischemic, and/orperi-infarct region of the myocardial tissue to promote expression ofSDF-1 from cells of the weakened, ischemic, and/or peri-infarct region.For example, agents that increase the transcription of a gene encodingSDF-1 increase the translation of an mRNA encoding SDF-1, and/or thosethat decrease the degradation of an mRNA encoding SDF-1 could be used toincrease SDF-1 protein levels. Increasing the rate of transcription froma gene within a cell can be accomplished by introducing an exogenouspromoter upstream of the gene encoding SDF-1. Enhancer elements, whichfacilitate expression of a heterologous gene, may also be employed.

Other agents can include other proteins, chemokines, and cytokines, thatwhen administered to the target cells can upregulate expression SDF-1form the weakened, ischemic, and/or peri-infarct region of themyocardial tissue. Such agents can include, for example: insulin-likegrowth factor (IGF)-1, which was shown to upregulate expression of SDF-1when administered to mesenchymal stem cells (MSCs) (Circ. Res. 2008,November 21; 103(11):1300-98); sonic hedgehog (Shh), which was shown toupregulate expression of SDF-1 when administered to adult fibroblasts(Nature Medicine, Volume 11, Number 11, November 23); transforminggrowth factor β (TGF-β); which was shown to upregulate expression ofSDF-1 when administered to human peritoneal mesothelial cells (HPMCs);IL-1β, PDGF, VEGF, TNF-α, and PTH, which are shown to upregulateexpression of SDF-1, when administered to primary human osteoblasts(HOBs) mixed marrow stromal cells (BMSCs), and human osteoblast-likecell lines (Bone, 2006, April; 38(4): 497-508); thymosin β4, which wasshown to upregulate expression when administered to bone marrow cells(BMCs) (Curr. Pharm. Des. 2007; 13(31):3245-51; and hypoxia induciblefactor 1α (HIF-1), which was shown to upregulate expression of SDF-1when administered to bone marrow derived progenitor cells (Cardiovasc.Res. 2008, E. Pub.). These agents can be used to treat specificcardiomyopathies where such cells capable of upregulating expression ofSDF-1 with respect to the specific cytokine are present or administered.

The SDF-1 protein or agent, which causes increases, and/or upregulatesexpression of SDF-1, can be administered to the weakened, ischemic,and/or peri-infarct region of the myocardial tissue neat or in apharmaceutical composition. The pharmaceutical composition can providelocalized release of the SDF-1 or agent to the cells of the weakened,ischemic, and/or peri-infarct region being treated. Pharmaceuticalcompositions in accordance with the application will generally includean amount of SDF-1 or agent admixed with an acceptable pharmaceuticaldiluent or excipient, such as a sterile aqueous solution, to give arange of final concentrations, depending on the intended use. Thetechniques of preparation are generally well known in the art asexemplified by Remington's Pharmaceutical Sciences, 16th Ed. MackPublishing Company, 1980, incorporated herein by reference. Moreover,for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biological Standards.

The pharmaceutical composition can be in a unit dosage injectable form(e.g., solution, suspension, and/or emulsion). Examples ofpharmaceutical formulations that can be used for injection includesterile aqueous solutions or dispersions and sterile powders forreconstitution into sterile injectable solutions or dispersions. Thecarrier can be a solvent or dispersing medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, liquidpolyethylene glycol, and the like), dextrose, saline, orphosphate-buffered saline, suitable mixtures thereof and vegetable oils.

Proper fluidity can be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Nonaqueousvehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, cornoil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, may also be used as solvent systems for compoundcompositions.

Additionally, various additives, which enhance the stability, sterility,and isotonicity of the compositions, including antimicrobialpreservatives, antioxidants, chelating agents, and buffers, can beadded. Prevention of the action of microorganisms can be ensured byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. In many cases, it willbe desirable to include isotonic agents, for example, sugars, sodiumchloride, and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin. According tomethods described herein, however, any vehicle, diluent, or additiveused would have to be compatible with the compounds.

Sterile injectable solutions can be prepared by incorporating thecompounds utilized in practicing the methods described herein in therequired amount of the appropriate solvent with various amounts of theother ingredients, as desired.

Pharmaceutical “slow release” capsules or “sustained release”compositions or preparations may be used and are generally applicable.Slow release formulations are generally designed to give a constant druglevel over an extended period and may be used to deliver the SDF-1 oragent. The slow release formulations are typically implanted in thevicinity of the weakened, ischemic, and/or peri-infarct region of themyocardial tissue.

Examples of sustained-release preparations include semipermeablematrices of solid hydrophobic polymers containing the SDF-1 or agent,which matrices are in the form of shaped articles, e.g., films ormicrocapsule. Examples of sustained-release matrices include polyesters;hydrogels, for example, poly(2-hydroxyethyl-methacrylate) orpoly(vinylalcohol); polylactides, e.g., U.S. Pat. No. 3,773,919;copolymers of L-glutamic acid and γ ethyl-L-glutamate; non-degradableethylene-vinyl acetate; degradable lactic acid-glycolic acid copolymers,such as the LUPRON DEPOT (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate); andpoly-D-(−)-3-hydroxybutyric acid.

While polymers, such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated, SDF-1 orthe agent can remain in the body for a long time, and may denature oraggregate as a result of exposure to moisture at 37° C., thus reducingbiological activity and/or changing immunogenicity. Rational strategiesare available for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism involves intermolecular S—S bondformation through thio-disulfide interchange, stabilization is achievedby modifying sulfhydryl residues, lyophilizing from acidic solutions,controlling moisture content, using appropriate additives, developingspecific polymer matrix compositions, and the like.

In certain embodiments, liposomes and/or nanoparticles may also beemployed with the SDF-1 or agent. The formation and use of liposomes isgenerally known to those of skill in the art, as summarized below.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

Phospholipids can form a variety of structures other than liposomes whendispersed in water, depending on the molar ratio of lipid to water. Atlow ratios, the liposome is the preferred structure. The physicalcharacteristics of liposomes depend on pH, ionic strength and thepresence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

Liposomes interact with cells via four different mechanisms: Endocytosisby phagocytic cells of the reticuloendothelial system such asmacrophages and neutrophils; adsorption to the cell surface, either bynonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. Varying the liposome formulation can alter whichmechanism is operative, although more than one may operate at the sametime.

Nanocapsules can generally entrap compounds in a stable and reproducibleway. To avoid side effects due to intracellular polymeric overloading,such ultrafine particles (sized around 0.1 μm) should be designed usingpolymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use in the methods, and such particles may be areeasily made.

For preparing pharmaceutical compositions from the compounds of theapplication, pharmaceutically acceptable carriers can be in any form(e.g., solids, liquids, gels, etc.). A solid carrier can be one or moresubstances, which may also act as diluents, flavoring agents, binders,preservatives, and/or an encapsulating material.

The following examples are for the purpose of illustration only and arenot intended to limit the scope of the claims, which are appendedhereto.

EXAMPLES Example 1

Stromal cell-derived factor-1 or SDF-1 is a naturally-occurringchemokine whose expression is rapidly upregulated in response to tissueinjury. SDF-1 induction stimulates a number of protectiveanti-inflammatory pathways, causes the down regulation ofpro-inflammatory mediators (such as MMP-9 and IL-8), and can protectcells from apoptosis. Furthermore, SDF-1 is a strong chemoattractant oforgan specific and bone marrow derived stem cells and progenitor cellsto the site of tissue damage, which promotes tissue preservation andblood vessel development. Based on observations that increasedexpression of SDF-1 led to improved cardiac function in ischemic animalmodels, we focused on developing a non-viral, naked-DNA SDF-1-encodingplasmid for treatment of ischemic cardiovascular disease. During thecourse of development, the plasmid was optimized based on cell cultureand small animal study results described below. The plasmid ACL-01110Skwas selected based on its ability to express transgenes in cardiactissue and to consistently improve cardiac function in pre-clinicalanimal models of ischemic cardiomyopathy. SDF-1 transgene expression inACL-01110Sk is driven by the CMV enhancer/promoter, CMV-intron A, andthe RU5 translational enhancer. The drug product, JVS-100 (formerlyACRX-100), is composed of plasmid ACL-01110Sk in 5% dextrose.

Initial studies in a rat model of heart failure demonstrated thatACL-01110S (an SDF-1 expressing precursor to ACL-01110Sk) improvedcardiac function after injection of the plasmid directly into theinfarct border zone of the rat hearts four weeks following an MI.Benefits were sustained for at least 8-10 weeks post-injection andcorrelated with increased vasculogenesis in the ACL-01110S treatedanimals. ACL-01110S was modified to optimize its expression profile.

Plasmid Dose-Dependent Expression in a Rat Model of MI

To determine the plasmid dose per injection that would provide maximalexpression in rat cardiac tissue, escalating doses (10, 50, 100, 500 μg)of the ACL-00011L luciferase plasmid were injected into infarcted rathearts. Lewis rats were subjected to a median sternotomy and the leftanterior descending artery (LAD) was permanently ligated, and injectedperi-MI at one site with 100 μl ACL-00011L plasmid in PBS. Whole bodyluciferase expression was measured in each dose cohort (n=3) bynon-invasive bioluminescent imaging (Xenogen, Hopkinton, Mass.) atbaseline and at 1, 2, 3, 4, and 5 days post-injection. The peakexpression increased up to a dose of 100 μg and saturated at higherdoses. Based on this dose-response curve, a dose of 100 μg wasdetermined to be sufficient for maximal plasmid expression in rathearts. ACL-00011L expressed the luciferase gene from a vector backboneequivalent to that used in construction of ACL-00011S, which expressesSDF-1.

Comparison of Cardiac Vector Expression in a Rat Model of Ischemic HeartFailure

The luciferase expressing equivalents of several SDF-1 plasmidcandidates were tested for expression in cardiac tissue in a rat modelof myocardial infarct (MI). Plasmid candidates differed in the promotersdriving expression and presence of enhancer elements. Lewis rats weresubjected to a median sternotomy and the left anterior descending artery(LAD) was permanently ligated and the chest was closed. Four weekslater, the chest was reopened, and the luciferase expressing plasmidswas directly injected (100 μg in 100 μl per injection) into 4peri-Myocardial infarction sites. At 1, 2, 4, 6, 8, and 10 dayspost-injection (and every 3-4 days following), rats were anesthetized,injected with luciferin and imaged with a whole-body Xenogen Luciferaseimaging system.

The two CMV driven plasmids tested, ACL-00011L and ACL-01110L yieldeddetectable luciferase expression within 24 hours of injection with aninitial peak of expression at 2 days post-injection.

ACL-01110L peak expression was 7 times greater than ACL-00011L andexpression was approximately 10 days longer (lasting up to 16 days postinjection). In contrast, ACL-00021L (αMHC driven plasmid) showed noinitial peak, but expressed at a low-level through day 25post-injection. These results support previous studies demonstratingthat CMV driven plasmids can be used for localized, transient proteinexpression in the heart and that the timeframe of therapeutic proteinexpression can be modulated through the inclusion of enhancer elements.

Efficacy of SDF-1 Plasmids in Rat Model of MI

SDF-1-encoding plasmids were tested in a rat model of MI to determine iffunctional cardiac benefit could be achieved. Lewis rats were subjectedto a median sternotomy and the LAD was permanently ligated immediatelydistal to the first bifurcation. Four weeks later, the chest wasreopened, and one of three SDF-1 expressing plasmids (ACL-01110S,ACL-00011S, or ACL-00021S) or saline was injected (100 μg per 100 μlinjection) into 4 peri-MI sites:

At baseline (pre-injection), and 2, 4, and 8 weeks post-injection, ratswere anesthetized and imaged with M-mode echocardiography. LVEF,fractional shortening, and LV dimensions were measured by a trainedsonographer who was blinded to randomization.

A strong trend in improvement in cardiac function was observed with bothCMV driven plasmids, ACL-01110S and ACL-00011S, compared to salinecontrols. ACL-01110S elicited a statistically significant increase infractional shortening at four weeks that was sustained 8 weeks afterinjection. In contrast, no difference in function was observed betweenαMHC driven plasmid ACL-00021S and saline. Furthermore, compared tocontrol, the ACL-01110S and the ACL-00011S-treated animals hadsignificant increases in large vessel density (ACL-01110S: 21±1.8vessels/mm²; ACL-00011S: 17±1.5 vessels/mm²; saline: 6±0.7 vessels/mm²,p<0.001 for both vs. saline) and reduced infarct size (ACL-01110S:16.9±2.8%; ACL-00011S: 17.8±2.6%; saline: 23.8±4.5%). Importantly,treatment with ACL-01110S demonstrated the largest improvement incardiac function and vasculogenesis, and caused the largest reduction ininfarct size.

In summary, in a rat model of ischemic heart failure, bothSDF-1-encoding plasmids driven by a CMV promoter provided functionalcardiac benefit, increased vasculogenesis, and reduction in infarct sizecompared to saline treatment. In all parameters tested, ACL-01110Sprovided the most significant benefit.

Transfection Efficiency of ACL-01110Sk and ACl-01010Sk in H9C2 Cells

In vitro transfection of H9C2 myocardial cells without transfectionreagents (i.e., —naked plasmid DNA was added to cells in culture) wereused to estimate in vivo transfection efficiencies of GFP versions ofJuventas lead plasmid vectors, ACL-01110Sk and ACL-01010Sk. H9C2 cellswere cultured in vitro and various amounts of pDNA (0.5 μg, 2.0 μg, 4.0μg, 5.0 μg) were added in 5% dextrose. The GFP vectors were constructedfrom the ACL-01110Sk (ACL-01110G) or ACL-01010Sk (ACL-01010G) backbones.At Day 3 post-transfection, GFP fluorescence was assessed by FACS toestimate transfection efficiency. The transfection efficiencies for theACL-01110G and ACL-01010G vectors in 5% dextrose ranged from 1.08-3.01%.At each amount of pDNA tested, both vectors had similar in vitrotransfection efficiencies. We conclude that the 1-3% transfectionefficiency observed in this study is in line with findings from previousstudies demonstrating a similar level of in vivo transfectionefficiency. Specifically, JVS-100 will transfect a limited butsufficient number of cardiac cells to produce therapeutic amounts ofSDF-1.

Example 2 Expression of Plasmid in Porcine Myocardium

A porcine occlusion/reperfusion MI model of the left anterior descendingartery (LAD) was selected as an appropriate large animal model to testthe efficacy and safety of ACRX-100. In this model, 4 weeks recovery isgiven between MI and treatment to allow time for additional cardiacremodeling and to simulate chronic ischemic heart failure.

Surgical Procedure

Yorkshire pigs were anesthesitzed and heparanized to an activatedclotting time (ACT) of ≧300 seconds, and positioned in dorsalrecumbency. To determine the contour of the LV, left ventriculographywas performed in both the Anterior—Posterior and Lateral views.

Delivery of Luciferase Plasmid into Porcine Myocardium

A deflectable guide catheter device was advanced to the left ventricleretrograde across the aortic valve, the guide wire was removed, and anLV endocardial needle injection catheter was entered through the guidecatheter into the LV cavity. Luciferase plasmid was injected at 4 sitesat a given volume and concentration were made into either the septal orlateral wall of the heart. Five combinations of plasmid concentration(0.5, 2, or 4 mg/ml) and site injection volumes (0.2, 0.5, 1.0 ml) weretested. Plasmid at 0.5 mg/ml was buffered in USP Dextrose, all otherswere buffered in USP Phosphate Buffered Saline. For each injection, theneedle was inserted into the endocardium, and the gene solution wasinjected at a rate of 0.8-1.5 ml/minute. Following injection, the needlewas held in place for 15 seconds and then withdrawn. After injectionswere completed, all instrumentation was removed, the incision wasclosed, and the animal was allowed to recover.

Harvesting of Myocardial Tissue

On Day 3 post injection, the animals were submitted to necropsy.Following euthanasia, the heart was removed, weighed, and perfused withLactate Ringers Solution until clear of blood. The LV was opened and theinjection sites identified. A 1 cm square cube of tissue was takenaround each injection site. Four (4) cubes harvested from the posteriorwall remote from any injection sites served as negative controls. Thetissue samples were frozen in liquid nitrogen and stored at −20 to −70°C.

Assessment of Luciferase Expression

The tissue samples were thawed and placed in a 5 ml glass tube. Lysisbuffer (0.5-1.0 ml) was added and tissue was disrupted using Polytronhomogenization (model PT1200) on ice. Tissue homogenate was centrifugedand protein concentration of the supernatant was determined for eachtissue sample using the Bio-rad Detergent-Compatible (DC) protein assayand a standard curve of known amounts of bovine serum albumin (BSA).Tissue sample homogenate (1-10 μl) was assayed using the Luciferaseassay kit (Promega).

The results of the experiment are shown in FIG. 1. The data shows thatexpression of the vector increases with increasing injection volume andincreasing concentration of DNA.

Example 3 Improvement in Cardiac Function by SDF-1 Plasmid Treatment inPorcine Model of Ischemic Cardiomyopathy Induction of MyocardialInfarction

Yorkshire pigs were anesthesitzed and heparanized to an activatedclotting time (ACT) of ≧250 seconds, and positioned in dorsalrecumbency. A balloon catheter was introduced by advancing it through aguide catheter to the LAD to below the first major bifurcation of theLAD. The balloon was then inflated to a pressure sufficient to ensurecomplete occlusion of the artery, and left inflated in the artery for90-120 minutes. Complete balloon inflation and deflation was verifiedwith fluoroscopy. The balloon was then removed, the incision was closed,and the animal was allowed to recover.

Enrollment Criteria

One month post-MI, cardiac function in each pig was assessed byechocardiography. If the LVEF was less than 40% and the LVESV wasgreater than 56.7 ml, the pig was enrolled in the study.

Surgical Procedure

Each enrolled pig was anesthesitzed and heparanized to an activatedclotting time (ACT) of ≧300 seconds, and positioned in dorsalrecumbency. To determine the contour of the LV, left ventriculographywas performed in both the Anterior—Posterior and Lateral views.

Delivery of SDF-1 Plasmid (ACL-01110Sk) into Myocardium

Each pig was randomized to one of 3 sacrifice points: 3 days, 30 days,or 90 days post-treatment, and to one of four treatment groups: control(20 injections, buffer only), low (15 injections, 0.5 mg/ml), mid (15injections, 2.0 mg/ml), or high (20 injections, 5.0 mg/ml). All plasmidwas buffered in USP Dextrose. The injection procedure is describedbelow.

A deflectable guide catheter device was advanced to the left ventricleretrograde across the aortic valve, the guide wire was removed, and anLV endocardial needle injection catheter was entered through the guidecatheter into the LV cavity. SDF-1 plasmid or buffer at randomized dosewas loaded into 1 ml syringes that were connected to the catheter. Eachinjection volume was 1.0 ml. For each injection, the needle was insertedinto the endocardium, and the solution was injected over 60 seconds.Following injection, the needle was held in place for 15 seconds andthen withdrawn. After injections were completed, all instrumentation wasremoved, the incision was closed, and the animal was allowed to recover.

At sacrifice, samples of tissues from the heart and other major organswere excised and flash frozen for PCR and histopathological analysis.

Assessment of Cardiac Function

Each animal had cardiac function assessed by standard 2-dimensionalechocardiography at day 0, 30, 60, and 90 post-injection (or untilsacrifice). Measurements of left ventricular volume, area, and wallmotion score were made by an independent core laboratory. The efficacyparameters measured are shown below in Table 1.

TABLE 1 Echocardiographic Parameters Variable Name Definition LVESV EndSystolic Volume measured in parasternal long-axis view LVEDV EndDiastolic Volume measured in parasternal long-axis view LVEF (LVEDV −LVESV)/LVEDV *100% WMSI Average of all readable wall motion scores basedon ASE 17 segment model and scoring system of 0-5.

The impact of SDF-1 plasmid on functional improvement is shown in FIGS.2-5. FIGS. 2-4 show that the low and mid doses of SDF-1 plasmid improveLVESV, LVEF, and Wall Motion Score Index at 30 days post-injectioncompared to control; whereas, the high dose does not show benefit. FIG.5 demonstrates that the cardiac benefit in the low and mid dose issustained to 90 days, as both show a marked attenuation in pathologicalremodeling, that is, a smaller increase in LVESV, compared to control.

Assessment of Vasculogenesis

Animals that were sacrificed at 30 days were assessed for vessel densityin the left ventricle using 7 to 9 tissue samples harvested from eachformalin-fixed heart. Genomic DNA was extracted and efficiently purifiedfrom formalin fixed tissue sample using a mini-column purificationprocedure (Qiagen). Samples from SDF-1 treated and control animals weretested for presence of plasmid DNA by quantitative PCR. Three to fivetissue samples found to contain copies of plasmid DNA at least 4-foldabove background (except in control animals) for each animal were usedto prepare slides and immunostained with isolectin. Cross-sections wereidentified and vessels counted in 20-40 random fields per tissue. Thevessels per field were converted to vessels/mm² and were averaged foreach animal. For each dose, data is reported as the average vessels/mm²from all animals receiving that dose.

FIG. 6 shows that both doses that provided functional benefit alsosignificantly increase vessel density at 30 days compared to control. Incontrast, the high dose, which did not improve function, did notsubstantially increase vessel density. This data provides a putativebiologic mechanism by which SDF-1 plasmid is improving cardiac functionin ischemic cardiomyopathy.

Biodistribution Data

JVS-100 distribution in cardiac and non-cardiac tissues was measured 3,30 and 90 days after injection in the pivotal efficacy and toxicologystudy in the pig model of MI. In cardiac tissue, at each time point,average JVS-100 plasmid concentration increased with dose. Art eachdose, JVS-100 clearance was observed at 3, 30 and 90 days followinginjection with approximately 99.999999% cleared from cardiac tissue atDay 90. JVS-100 was distributed to non-cardiac organs with relativelyhigh blood flow (e.g. heart, kidney, liver, and lung) with the highestconcentrations noted 3 days following injection. JVS-100 was presentprimarily in the kidney, consistent with renal clearance of the plasmid.There were low levels of persistence at 30 days and JVS-100 wasessentially undetectable in non-cardiac tissues at 90 days.

Conclusions

Treatment with JVS-100 resulted in significantly increased blood vesselformation and improved heart function in pigs with ischemic heartfailure following a single endomyocardial injection of 7.5 and 30 mg.The highest dose of JVS-100 tested (100 mg) showed a trend in increasedblood vessel formation but did not show improved heart function. None ofthe doses of JVS-100 were associated with signs of toxicity, adverseeffects on clinical pathology parameters or histopathology. JVS-100 wasdistributed primarily to the heart with approximately 99.999999% clearedfrom cardiac tissue at 90 days following treatment. JVS-100 wasdistributed to non-cardiac organs with relatively high blood flow (e.g.,heart, kidney, liver, and lung) with the highest concentrations in thekidneys 3 days following injection. JVS-100 was essentially undetectablein the body 90 days after injection with only negligible amounts of theadministered dose found in non-cardiac tissues. Based on these findingsthe no observed adverse effect level (NOAEL) for JVS-100 in the pigmodel of MI was 100 mg administered by endomyocardial injection.

Example 4 Porcine Exploratory Study: LUC Injections by TransarterialInjection in Chronic MI Pigs Methods

One pig with a previous LAD occlusion/reperfusion MI and an EF>40%, wasinjected with ACL-01110Sk with a Transarterial catheter. Two injectionsin the LAD and 2 in the LCX were performed with an injection volume of2.5 ml and a total injection time of 125-130 sec. One additionalinjection in the LCX of 3.0 ml with a total injection time of 150 secwas performed with conttast mixed with the plasmid.

Sacrifice and Tissue Collection

Three days following the injections, the animal was euthanized. Aftereuthanasia, the heart was removed, drained of blood, placed on an icecold cutting board and further dissected by the necropsy technician orpathologist. The non-injected myocardium from the septum was obtainedvia opening the right ventricle. The right ventricle was trimmed fromthe heart and placed in cold cardioplegia. New scalpel blades were usedfor each of the sections.

Next, the left ventricle was opened and the entire left ventricle wasexcised by slicing into 6 sections cutting from apex to base. The LV wasevenly divided into 3 slices. Following excision, each section was ableto lay flat. Each section (3 LV sections, 1 RV section, and 1 pectoralmuscle) was placed in separate labeled containers with cold cardioplegiaon wet ice, and transported for luciferase analysis.

Luciferase Imaging

All collected tissues were immersed in luciferin and imaged with aXenogen imaging system to determine plasmid expression.

Results

A representative image of the heart is shown in FIG. 8. The coloredspots denote areas of luciferase expression. These spots showed RelativeLight Units (RLUs) of greater than 10⁶ units, more than 2 orders ofmagnitude above background. This data demonstrated that the catheterdelivered plasmid sufficient to generate substantial plasmid expressionover a significant portion of the heart.

Example 5 Clinical Study Example

Ascending doses of JVS-100 are administered to treat HF in subjects withischemic cardiomyopathy. Safety is tracked at each dose by documentingall adverse events (AEs), with the primary safety endpoint being thenumber of major cardiac AEs at 30 days. In each cohort, subjects willreceive a single dose of JVS-100. In all cohorts, therapy efficacy isevaluated by measuring the impact on cardiac function via standardechocardiography measurements, cardiac perfusion via Single PhotonEmission Computed Tomography (SPECT) imaging, New York Heart Association(NYHA) class, six minute walk distance, and quality of life.

All subjects have a known history of systolic dysfunction, prior MI, andno current cancer verified by up to date age appropriate cancerscreening. All subjects are screened with a physician visit, and acardiac echocardiogram. Further baseline testing such as SPECT perfusionimaging, is performed. Each subject receives fifteen (15) 1 mlinjections of JVS-100 delivered by an endocardial needle catheter tosites within the infarct border zone. Three cohorts (A, B, C) will bestudied. As shown in Table 2, dose will be escalated by increasing theamount of DNA per injection site while holding number of injection sitesconstant at 15 and injection volume at 1 ml. Subjects are monitored forapproximately 18 hours post-injection and have scheduled visits at 3 and7 days post-injection to ensure that there are no safety concerns. Thepatient remains in the hospital for 18 hours after the injection toensure all required blood collections (i.e., cardiac enzymes, plasmaSDF-1 protein levels) are performed. All subjects have follow-up at 30days (1 month), 120 days (4 months), and 360 days (12 months) to assesssafety and cardiac function. The primary safety endpoint are majoradverse cardiac events (MACE) within 1 month post-therapy delivery. AEswill be tracked for each subject throughout the study. The followingsafety and efficacy endpoints will be measured:

Safety:

Number of Major Adverse Cardiac Events (MACE) at 30 days post-injection

Adverse Events throughout the 12 month follow-up period

Blood lab Analysis (Cardiac Enzymes, CBC, ANA)

SDF-1 Plasma Levels

Physical assessment

Echocardiography

AICD monitoring

ECG

Efficacy:

Change from baseline in LVESV, LVEDV, LVEF, and wall motion score index

Change from baseline in NYHA classification and quality of life

Change from baseline in perfusion as determined by SPECT imaging

Change from baseline in Six Minute Walk Test distance

TABLE 2 Clinical Dosing Schedule # of Amount of Injection # InjectionTotal Dose Cohort Subjects DNA/site volume/site Sites of DNA Cohort A 40.33 mg  1.0 ml 15  5 mg Cohort B 6 1.0 mg 1.0 ml 15 15 mg Cohort C 62.0 mg 1.0 ml 15 30 mg

Based on preclinical data, delivery of JVS-100 is expected to elicit animprovement cardiac function and symptoms at 4 months that sustains to12 months. At 4 months following JVS-100 injection, compared to baselinevalues, an improvement in six minute walk distance of about greater than30 meters, an improvement in quality of life score of about 10%, and/oran improvement of approximately 1 NYHA class are anticipated. Similarly,we expect a relative improvement in LVESV, LVEF, and/or WMSI ofapproximately 10% compared to baseline values.

Comparative Example 1

Evaluation of Cardiac Function by Echocardiography in Chronic HeartFailure Pies after Treatment with ACL-01110Sk or ACL-01010Sk

Purpose

The purpose of this study is to compare functional cardiac response toSDF-1 plasmids ACL-01110Sk or ACL-01010Sk after endomyocardial catheterdelivery in a porcine model of ischemic heart failure

This study compared efficacy of ACL-01110Sk and ACL-01010Sk in improvingfunction in a porcine ischemic heart failure model. In this study, theplasmids were delivered by an endoventricular needle injection catheter.Efficacy was assessed by measuring the impact of the therapy on cardiacremodeling (i.e., left ventricular volumes) and function (i.e., leftventricular ejection fraction (LVEF)) via echocardiography.

Methods

Briefly, male Yorkshire pigs were given myocardial infarctions by LADocclusion via balloon angioplasty for 90 minutes. Pigs having anejection fraction <40% as measured by M-mode echocardiography 30 dayspost-infarct were enrolled. Pigs were randomized to one of 3 groups tobe injected with either Phosphate Buffered Saline (PBS, control),ACL-01110Sk in PBS, or ACL-01010Sk in PBS using an endoventricularneedle injection catheter delivery system (Table 3).

TABLE 3 Initial Study Design: SDF-1 Therapy for Chronic Heart Failure inPigs Injection # In- # of volume/ Amount of jection Total Group PlasmidPigs site DNA/site Sites DNA 1 Vehicle 3 200 μl N/A 10 n/a 2 ACL-01010Sk3 200 μl 400 μg 10 4 mg 3 ACL-01110Sk 3 200 μl 400 μg 10 4 mg

Echocardiograms were recorded prior to injection and at 30 and 60 dayspost-injection. Table 8 below defines the variables as they are referredto in this report.

TABLE 4 Definition of variables Variable Name Definition LVESV EndSystolic Volume measured in parasternal long-axis view LVEDV EndDiastolic Volume measured in parasternal long-axis view LVEF (LVEDV −LVESV)/LVEDV *100%

Results

The baseline echocardiographic characteristics at time of initialinjection (Day 30 post-MI) for all enrolled animals in this report (n=9)as reported by the echocardiography core laboratory, are provided inTable 5 below.

TABLE 5 Baseline characteristics Baseline Value Baseline Value BaselineValue Parameter Group 1 Group 2 Group 3 LVESV  78 ± 18 ml 67 ± 2 ml  86± 31 ml LVEDV 132 ± 30 ml 114 ± 11 ml 130 ± 36 ml LVEF 41 ± 1%  41 ± 5% 34 ± 10%

Table 5 shows the LVESV, LVEF and LVEDV at 0 and 30 days post-initialinjection. Control PBS animals demonstrated an increase in LVESV andLVEDV and no improvement in LVEF consistent with this heart failuremodel. The treatment groups did not reduce cardiac volumes or increaseLVEF compared to control. Similar results were obtained at 60 dayspost-initial injection.

Comparative Example 2

A strategy to augment stem cell homing to the periinfarct region bycatheter-based transendocardial delivery of SDF-1 in a porcine model ofmyocardial infarction was investigated to determine if it would improveleft ventricular perfusion and function. The catheter-based approach hasbeen used successfully for cell transplantation and delivery ofangiogenic growth factors in humans.

Female German landrace pigs (30 kg) were used. After an overnight fast,animals were anesthetized and intubated.

A 7 French sheath was placed in the femoral artery with the animal in asupine position. An over-the-wire balloon was advanced to the distalLAD. The balloon was inflated with 2 atm and agarose beads were injectedslowly over 1 min via the balloon catheter into the distal LAD. After 1minute the balloon was deflated and the occlusion of the distal LAD wasdocumented by angiography. After induction of myocardial infarctionanimals were monitored for 3-4 h until rhythm and blood pressure wasstable. The arterial sheath was removed, carprofen (4 mg/kg) wasadministered intramuscularly and the animals were weaned from therespirator. Two weeks after myocardial infarction animals wereanesthetized. Electromechanical mapping of the left ventricle wasperformed via an 8F femoral sheath with the animal in the supineposition. After a complete map of the left ventricle had been obtained,human SDF-1 (Peprotec, Rocky-Hill, N.J.) was delivered by 18 injections(5 μg in 100 μml saline) into the infarct and periinfarct region via aninjection catheter. 5 μg per injection was used to adjust for thereported efficiency of the catheter injection. Injections were performedslowly over 20 s and only when the catheter's tip was perpendicular) tothe left ventricular wall, when loop stability was <2 mm and when needleprotrusion into the myocardium provoked ectopic ventricular extra beats.Control animals underwent an identical procedure with sham injections.Echocardiography excluded postinterventional pericardial effusion.

Twenty (20) animals completed the study protocol: 8 control animals and12 SDF-1 treated animals. For myocardial perfusion imaging only 6control animals could be evaluated due to technical problems. Infarctlocation was anteroseptal in all animals.

Infarct size in percent of the left ventricle as determined bytetrazolium staining was 8.9±2.6% in the control group and 8.9±1.2% inthe SDF-1 group. Left ventricular muscle volume was similar in bothgroups (83±14 ml versus 95±10 ml, p=ns). Immunofluorescence stainingrevealed significantly more vWF-positive vessels in the peri-infarctarea in SDF-1 treated animals than in control animals (349±17/mm² vs.276±21/mm², p<0.05). A profound loss of collagen in the periinfarct areawas observed in SDF-1 treated animals as compared to control animals(32±5% vs. 61±6%, p<0.005). The number of inflammatory cells(neutrophils and macrophages) within the periinfarct area was similar inboth groups (332±51/mm² vs. 303±55/mm², p=ns). Global myocardialperfusion did not change from baseline to follow-up SPECT and there wasno difference between groups. Final infarct size was similar in bothgroups and compared well to the results of tetrazolium stainingSegmental analysis of myocardial perfusion revealed decreased traceruptake in apical and anteroseptal segments with significant differencesbetween myocardial segments. However, tracer uptake at baseline andfollow-up were nearly identical in control and SDF-1 treated animals.There were no differences in end diastolic and end systolic volumesbetween groups. However, stroke volume increased in control animals anddecreased slightly in SDF-1 treated animals. The difference between bothgroups was significant.

Similarly, ejection fraction increased in control animals and decreasedin SDF-1 treated animals. The difference between groups showed a strongtrend (p=0.05). Local shortening, another parameter of ventricularmechanical function, did not change in control animals. However, localshortening decreased significantly in SDF-1 treated animals, resultingin a significant difference between groups. There were no significantdifferences in unipolar voltage within and between groups. Significantcorrelations between baseline ejection fraction and stroke volume andbaseline local shortening (EF and LS: r=0.71, SV and LS: r=0.59) werenoted. Similar results were obtained for follow-up values (EF and LS:r=0.49, SV and LS: r=0.46). The change in local shortening correlatedsignificantly with the change in ejection fraction (r=0.52) and strokevolume (r=0.46). There was neither a correlation between localshortening and enddiastolic volume (baseline r=−0.03, follow-up r=0.12)nor between ejection fraction and enddiastolic volume (baseline r=−0.04,follow-up r=0.05). Segmental analysis of EEM data showed decreasedunipolar voltage and local shortening in the anteroseptal segments withsignificant differences between myocardial segments at baseline. Thedistribution of unipolar voltage values in myocardial segments wassimilar in both groups at baseline and at follow-up. Segmental localshortening did not change in the control group. However, it decreased inthe SDF-1 group, mainly due to a decrease in the lateral and posteriorsegment of the left ventricle. There was a significant interactionbetween assignment to SDF-1 and follow-up vs. baseline.

The study described above demonstrated that a single application ofSDF-1 protein was insufficient to produce functional cardiac benefit.

From the above description of the application, those skilled in the artwill perceive improvements, changes and modifications. Suchimprovements, changes and modifications within the skill of the art areintended to be covered by the appended claims. All patents, patentapplications and publications cited herein are incorporated by referencein their entirety.

What is claimed:
 1. An SDF-1 plasmid comprising an SDF-1α cDNA sequencehaving its expression driven by a CMV enhancer and promoter, CMV-intronA and the RU5 translational enhancer.
 2. The SDF-1 plasmid according toclaim 1, wherein said plasmid comprises, in a 5′ to 3′ order, nucleotidesequences for a CMV enhancer, a CMV promoter, CMV-intron A, RU5, SDF-1α,BGH polyA, and ColE1 origin.
 3. The SDF-1 plasmid according to claim 2,wherein said SDF-1 plasmid is plasmid ACL-01110Sk.
 4. A preparationcomprising an SDF-1 plasmid according to claim 1 and a pharmaceuticallyacceptable carrier.
 5. The preparation according to claim 4, whereinsaid preparation is injectable.
 6. The injectable preparation accordingto claim 5, wherein said pharmaceutically acceptable carrier is 5%dextrose.
 7. The injectable preparation according to claim 5, comprisingfrom about 0.33 mg/ml to about 5 mg/ml of said SDF-1 plasmid.
 8. Theinjectable preparation according to claim 7, comprising about 2 mg/ml ofsaid SDF-1 plasmid.
 9. A method of treating an ischemic cardiomyopathyin a mammal comprising: administering to a weakened, ischemic, orperi-infarct region of the myocardium, by injection, a solutioncomprising from about 5 mg to about 100 mg of the plasmid of claim 1.10. The method of claim 9, wherein the SDF-1 plasmid is administered tothe weakened, ischemic, or peri-infarct region in multiple injections ofthe solution with each injection comprising from about 0.33 mg/ml toabout 5 mg/ml of SDF-1 plasmid.
 11. The method of claim 10, wherein eachinjection has a volume of at least 0.2 ml.
 12. The method of claim 11,wherein the SDF-1 plasmid is administered to the weakened, ischemic, orperi-infarct region in at least 10 injections.
 13. The method of claim12, wherein the amount of SDF-1 plasmid administered to the weakened,ischemic, or peri-infarct region is about 5 mg.
 14. The method of claim12, wherein the volume of solution of SDF-1 plasmid administered to theweakened, ischemic, or peri-infarct region is about 10 ml.
 15. Themethod of claim 9, wherein the SDF-1 plasmid is administered to theweakened, ischemic, or peri-infarct region in at least 10 injections ofthe solution with each injection comprising about 0.5 mg/ml to about 5mg/ml of SDF-1 plasmid/solution and each injection has a volume of about1.0 ml.
 16. The method of claim 9, wherein SDF-1 is expressed at atherapeutically effective amount in the weakened, ischemic, orperi-infarct region for greater than three days.
 17. The method of claim9, wherein the myocardial tissue of the subject is imaged to define thearea of weakened, ischemic, or peri-infarct region prior toadministration of the SDF-1 plasmid and the SDF-1 plasmid isadministered to the weakened, ischemic, or peri-infarct region definedby the imaging.
 18. The method of claim 17, wherein the imaging includesat least one of echocardiography, magnetic resonance imaging, coronaryangiogram, electroanatomical mapping, or fluoroscopy.
 19. The method ofclaim 11, wherein the SDF-1 plasmid is administered to the weakened,ischemic, or peri-infarct region in at least 15 injections.
 20. Themethod of claim 19, wherein the total amount of SDF-1 plasmidadministered to the weakened, ischemic, or peri-infarct region is fromabout 15 mg to about 30 mg.
 21. The method of claim 9, wherein thesolution comprising a plasmid encoding SDF-1 further comprises apharmaceutically acceptable carrier.
 22. The method of claim 21, whereinthe pharmaceutically acceptable carrier is dextrose.